Allergies are caused by a dysfunction in the immune system, which reacts to innocuous proteins contained in pollen, mites, epithelia and certain foods by producing IgE-class antibodies.
Recent data indicate that above 10% of the population in Western countries suffer from this disease, the symptoms of which may deteriorate with time giving rise to e.g. asthma or a sensitization to other allergens thus making more difficult the choice of the appropriate therapy.
Specific hyposensitizing immunotherapy (SIT), unlike pharmacological therapy, is the only etiological treatment of allergic diseases capable of favourably changing the immunological parameters characteristic of such diseases.
The hyposensitizing immunotherapy consists in the administration of increasing doses of standardized extracts (vaccines) obtained from the same substance which causes the disease (1). In this way, a sort of immunological tolerance to said substance is gradually induced in the patient with following disappearance of the allergic symptoms.
However, the risk of eliciting serious side effects (2), although remarkably reduced with the use of either slow-release vaccines or vaccines administered through routes alternative to injection, has in fact limited the application of specific hyposensitizing immunotherapy in the treatment of allergic diseases.
In recent years, most attention has been focused on the development of effective, safer vaccines, particularly vaccines consisting of mutagenized recombinant proteins, i.e. hypoallergenic variants capable of favourably influencing the natural progression of the disease without causing undesired side effects (3).
One of the beneficial factors of SIT is the induction if IgG antibodies specific for the sensitizing allergen. These (protective) antibodies can inhibit the antigen-IgE binding, specifically the IgE binding to Bet v 2 antigen, altering the molecule's tridimensional conformation (4,5). The development of vaccines containing hypoallergenic recombinant proteins with unaltered immunogenic properties may improve the therapeutic approach to allergic diseases.
The pollen of plants taxonomically known as Fagales (birch, alder, hazel, oak, hornbeam) is one of the most important causes of allergic rhinitis and asthma in the temperate regions. The two major allergens of birch pollen. Bet v 1 (cDNA deposited at GenBank acc. No. X15877) and Bet v 2 (acc. No. M65179) are proteins with molecular weight of 17 and 14 kD, respectively (6, 7). Bet v 2 belongs to the profilin family, which are ubiquitous cytoplasmic proteins involved in the regulation of eukaryotic cell cytoskeleton. They specifically interact with at least two cellular (macro)molecules, that is, phosphatidylinositol-4,5-bisphosphate, thereby preventing the hydrolysis of this fatty acid by C-γ phospholypase (8), and actin, modulating its polymerization (9). The high expression of profilins in mature and germinative pollen suggests their involvement in the regulation of the microfilament precursors which take part in the germination process (10). Profilins were identified as allergens in the pollen from many arboreous and herbaceous plants and in many fruits and vegetables and thus they were defined ‘pan-allergens’, despite the fact that they are found in only 20% of patients allergic to pollen (11, 12).
The high sequence homology, higher than 60% in most plant profilins of various origin, causes cross-sensitization not only with pollen from botanically correlated (13) and non-correlated (14) plants but also between pollen and plant aliments (15) or between pollen and latex (16). The homology between plant and mammalian profilins is rather low, nonetheless they proved able to bind actin from different species and showed interchangeability (17, 18, 19). An explanation is that all profilins share a similar tridimensional structure, as shown using X-ray crystallography (20, 21, 22).
Many studies confirm the immunologic equivalence of profilins. In fact it was shown that IgEs from patients sensitized to a determined profilin are able to bind profilins of different origin and that IgE binding to profilins can be mutually inhibited (16).
The high cross-reactivity between different profilins allows the use of a single profilin for allergy diagnosis and recombinant Bet v 2 is often used as the allergen of choice for profilin-specific IgE determination (23, 24).
There are many studies on the determination of profilin IgE epitopes.
Vrtala (1996) (25) mutagenized Bet v 2 at positions Phe44 and Gln47, which were changed in Tyr44, Glu47 and Asn47, according to contemporaneous studies carried out by the some group (26), where a linear epitope recognized by the monoclonal antibody 4A6 was identified. The epitope recognized by this antibody was mapped using synthetic dodecapeptides which spanned the entire amino acid sequence of Bet v2. The peptides which more efficiently bound the antibody contained the regions between amino acids 38-49 and 40-51. The importance of Gln47 residue in the IgG-peptide binding was supported by the evidence that 4A6 was not able to recognize the profilins from Nicotiana tabacum and Phleum pratense, whose sequences present a glutamate in place of Gln47 in Bet v 2. Unlike the Gln47→Glu47 mutation, the change from Phe44 to Tyr44 or from Gln47 to Asn47 did not affect the antibody binding. The same mutations (Gln47 to Glu or Asn and Phe44 to Tyr44) applied to recombinant Bet v 2 were unable to diminish the binding between profilin and IgE, as shown by immunoblotting and ELISA experiments (25).
In a subsequent study published in 1997 (22), the main IgE epitopes were identified by cloning random fragments of birch profilin cDNA from an expression library assayed with sera from patients allergic to profilins. Three regions, corresponding to the alpha helices located at the amino (aa 1-30) and carboxy (aa 106-132) termini and to a fragment comprised between residues 30 and 50, proved more reactive.
In a subsequent study (23), the search for IgE epitopes was based on the comparison between theoretical structural models for profilins from different plants and birch or latex profilin crystals. Eleven potential conformational epitopes were predicted, consisting of contiguous amino acid regions, at least 20% of which are surface-exposed. Two types of epitopes resulted from a comparison of the amino acid sequences with the conformational models: the species-specific ones, characterised by a high variability, and the highly conserved ones, which are more likely involved in the cross-reactivity between profilins from different plants. The amino acid sequence alignments reflect the results of Fedorov's study (22), evidencing two potential linear epitopes at the N-terminus, three in the region between residues 30 and 80 and additional two at the profilin C-terminus. All these regions are highly conserved in the plant profilins assessed in this study. The prediction of potential conformational epitopes was based on the analysis of a 3D model for the latex profilin Hev b 8. The analysis evidenced 12 protruding residues, selected as centres of the epitopes. Although all potential epitopes were conformational, they contained linear sequences and either conserved or variable residues. No test was reported in this study to confirm the specific IgE-binding capability of the proposed epitopes.
A more recent publication concerns the identification of IgE epitopes of Cucumis melo's profilin (27). By determining the IgE reactivity of peptides spanning the entire amino acid sequence of this protein, two linear epitopes were identified, which are strongly recognized by the serum of patients allergic to melon: E1, comprising residues 66-75 and 81-93, and E2 consisting of amino acids 95-99 and 122-131. Two additional epitopes were characterised by a weaker IgE response, namely E3 (residues 2-10) and E4 (35-45). The overlap of peptides corresponding to epitopes E1 and E2 with the melon profilin 3D model, indicates two regions with well-defined electrostatic properties: E1 and E2, which are associated with electropositive and electronegative protein domains, respectively.
The data available from the literature suggest the molecular portions where the IgE profilin epitopes are located but fail to indicate the amino acids involved in IgE binding.